Tissue engineering attempts to create three-dimensional tissue structures on which cells and other biomolecules may be incorporated. These structures or scaffolds guide the organization, growth and differentiation of cells in the process of forming functional tissue by providing physico-chemical cues. To successfully incorporate a scaffold within the host body depends on efficient communication between cells, tissues and the host system as a whole. The scaffolds and cells to be incorporated must interact with adhesion and growth factor receptors and the scaffold must eventually degrade.
Many disease conditions or injuries of the body require the repair or replacement of damaged tissues, but the body itself may not be able to replace or repair the tissue satisfactorily or within an appropriate time scale. Thus many methods of disease or injury treatment involve methods of augmenting the body's natural repair mechanisms and often rely on the use of implantable biological scaffolds or prostheses. Ideally an implantable prosthesis should be chemically inert, noncarcinogenic, capable of resisting mechanical stress, sterilisable and resistant to the actions of tissue fluids as well as being non-inflammatory and hypoallergenic.
A number of scaffolds are known. These scaffolds may be synthetic and/or biological in nature.
Biological scaffolds have a number of advantages in that communication with existing body cells is instantaneous, they undergo a natural process of degradation, and existing biological signals attenuate incorporated signals such as the growth factors and cytokines which are inherently present in these scaffolds. On the other hand, these scaffolds exhibit great variability in terms of their morphological and biological properties, they are complex to manufacture and susceptible to contamination. Additionally, modifications are not as easy compared to synthetic scaffolds. There are limitations such as size, shape and the properties that can be imparted to a biological scaffold. Biological scaffolds are subjected to a number of processing procedures such as sterilizing and cross-linking before implantation. These processing procedures may alter the innate characteristics of the scaffold. The best processing procedure will minimize the loss of natural scaffold-properties. Thus the minimally altered scaffold will retain most natural molecules required to establish the expected therapeutic properties of the tissue-engineered organ or tissue. Upon implantation, these retained natural molecules are also further amenable to natural degradation. The biomolecular architecture of a biological scaffold might not have been extensively studied and documented poorly compared to synthetic scaffolds. This ignorance is very often acceptable so long as the isolated scaffold continues to provide favourable or desired function over detrimental effects.
Synthetic scaffolds have the disadvantage that cell recognition is difficult, surface alterations such as mobilisation of growth factors to the scaffold (to permit cell migration, proliferation and adhesion) might be needed in some applications, they are subject to hydrolysis/enzymatic degradation and generate no biological signals. On the other hand they are consistent, easy to manufacture, easy to modify, and prototyping is easy.
One of the aims of tissue engineering is the production of tissue construct that can restore or repair lesions to a physiological status compatible with life. Identification of substrates, generally called scaffolds, for supporting growth and function of cells is therefore essential to successful tissue engineering. Various tissues and tissue components of animal origin are currently used as scaffolds. Xenogenic scaffolds have variable properties and still require optimisation of those properties before clinical use. The scaffolds derived from organs like small intestine, urinary bladder and oesophagus of pig, dog and sheep are available for clinical use as for example vascular grafts and skin grafts. However, the search for better scaffold substrates for tissue engineering applications is continuing. Although scaffolds derived from small intestine and urinary bladder can be used for certain applications, they are not suitable for universal use because of their defined range of mechanical and biological properties. Therefore there is continuing need to produce new scaffolds for specific clinical applications.
The present invention in one aspect utilizes transglutaminase in the production of a tissue scaffold. Transglutaminases are a class of natural enzymes that catalyse the acyl-transfer reaction between the ε-amino group of lysine and the γ-carboxyamide group of glutamine in proteins. Recent interest in transglutaminase can be attributed to the discovery of microbial transglutaminase (mTGase), (38) derived from a variant of Streptoverticillium mobaraense. mTGase is a calcium-independent enzyme that catalyses the formation of covalent crosslinks between glutamine and lysine residues in proteins. This enzyme has shown promise as a cross-linking agent (36-39) and has a wide range of applications, particularly in the food industry. The effective cross-linking of proteins by mTGase depends not solely on the presence of glutamine and lysine residues in the primary structure but also on the tertiary structure of the protein (40, 41).
In 1964 Curtis et al (2) proposed the concept that cells reacted to the topography upon which they were cultured, since then the study of cellular response to micro-topography has been substantial (3-8) and more recently cellular responses to nano-topography has also been investigated (9-13). Nano-scale topographic features modulate cell adhesion, spreading, focal adhesion formation, orientation, proliferation, differentiation and migration and are thought to have a more substantial influence on cellular behaviour than micro-scale features (10, 13-15). Cells evidently respond to topographical cues and are more likely to form a confluent, fully functional, physiologically similar layer on a topography with which they are familiar.
Several artificial and biological, biodegradable and non-biodegradable tissue engineering products are available to treat a range of tissues and locations throughout the body. Non-biodegradable membranes are prone to rejection in the short term and failure in the long term. Biodegradable membranes have been successful in eliciting a regeneration of body tissue that takes over from the implant as it dissolves. This healed area may be very different from the original tissue, because the prosthetic membrane does not elicit a proper healing response. Natural, acellular materials are being developed to in addition to current technologies to create a biological biodegradable scaffold. The primary structural material for these matrices is collagen but, because they are extracted directly from whole tissue, the signal pathways, enzymes, and growth factors naturally present in these tissues are still present. This sparks a natural healing response in the host tissue. Areas repaired with these materials tend to look and behave more like the original host tissue than ingrowth into inert biodegradable matrices.
Extracellular matrix (ECM) is the natural scaffold responsible for active tissue remodeling—in situ. Submucosa of hollow organs are connective tissues rich in ECM and have minimal cell content. Small intestinal submucosa (SIS) and urinary bladder submucosa are examples of such materials, which have been used as tissue engineering scaffolds with reasonable success.
As used herein the terms ‘extracellular matrix’ (ECM) is synonymous with the term ‘cholecyst-derived extracellular matrix’ (CEM) and similarly, ECM as used herein is defined as a tissue derived from the submucosal area of the cholecyst, except where the meaning(s) clearly refers to the extracellular matrix or ECM derived from a different tissue (for example, small intestine). In addition, the term ‘gallbladder’ is used synonymously with ‘cholecyst’.